Aneuploidy Underlies a Multicellular Phenotypic Switch

Aneuploidy underlies a multicellular phenotypic switch

Zhihao Tana,b,1, Michelle Haysa,1, Gareth A. Cromiea, Eric W. Jefferya, Adrian C. Scotta, Vida Ahyongc, Amy Sirra, Alexander Skupina,d, and Aimée M. Dudleya,b,2

aInstitute for Systems Biology, Seattle, WA 98109; bMolecular and Cellular Biology Program, University of Washington, Seattle, WA 98105; cTetrad Program, University of California, San Francisco, CA 94122; and dLuxembourg Center for Systems Biomedicine, University of Luxembourg, L-4362 Esch-sur-Alzette, Luxembourg

Edited by Angelika Amon, Massachusetts Institute of Technology, Cambridge, MA, and approved May 31, 2013 (received for review January 25, 2013)

Although microorganisms are traditionally used to investigate and wrinkled colonies: state changes that are associated with unicellular processes, the yeast Saccharomyces cerevisiae has the biochemically distinct capsular polysaccharides (18), changes in ability to form colonies with highly complex, multicellular struc- karyotype (18), and differential fungal burden in infected host tures. Colonies with the “fluffy” morphology have properties rem- organs (19). Because these different states may exhibit differ- iniscent of bacterial biofilms and are easily distinguished from the ential virulence, cell surface properties, or resistance to host im- “smooth” colonies typically formed by laboratory strains. We have mune defenses (2, 3), understanding the mechanisms underlying identified strains that are able to reversibly toggle between the phenotypic switching could prove essential for the development of fluffy and smooth colony-forming states. Using a combination of effective therapeutics against microbial pathogens. flow cytometry and high-throughput restriction-site associated Some strains of S. cerevisiae also exhibit a striking phenotypic DNA tag sequencing, we show that this switch is correlated with switch. Although most laboratory strains produce relatively un- a change in chromosomal copy number. Furthermore, the gain of structured “smooth” colonies, some natural isolates of S. cer- a single chromosome is sufficient to switch a strain from the fluffy evisiae produce colonies with complex multicellular features, to the smooth state, and its subsequent loss to revert the strain such as folds, crevices, and channels that form as the colony back to the fluffy state. Because copy number imbalance of six of grows on solid media (20, 21). These “fluffy” colonies possess the 16 S. cerevisiae chromosomes and even a single gene can mod- properties similar to those of microbial biofilms, including the ulate the switch, our results support the hypothesis that the state secretion and maintenance of an extracellular matrix (21–23), switch is produced by dosage-sensitive genes, rather than a general localized expression of drug efflux pumps (23), increased ad- response to altered DNA content. These findings add a complex, herence (24), and the use of cell–cell communication (25). Fluffy multicellular phenotype to the list of molecular and cellular traits colony formation requires the function and coordination of nu- known to be altered by aneuploidy and suggest that chromosome merous pathways that underlie the trait, and the deletion of key missegregation can provide a quick, heritable, and reversible mech- factors produces a smooth colony phenotype (26–28). Fluffy anism by which organisms can toggle between phenotypes. colonies may benefit from both rapid acquisition of larger ter- ritories as well as an increased capacity for nutrient and water colony morphology | copy number variation | phenotypic switching | transport (21). Interestingly, some fluffy strains can switch to the bet-hedging smooth morphology at a rate significantly higher than that expected for spontaneous mutations (21, 29). henotypic switching allows cells or organisms to reversibly Results Ptoggle between multiple, heritable states (1–3). These phenotypic states can confer distinct properties to cells that may be Bidirectional Toggling Between Phenotypic States. While studying advantageous in some environments, e.g., increased resistance to colony morphology in the haploid progeny of a cross between a Japanese sake strain and an Ethiopian white tecc strain (SI heat (4), faster proliferation rates in the presence of excess − Appendix 3

, Table S1), we observed a high frequency (10 )ofcol- GENETICS nutrients (5), or altered interaction with host immune systems fl (6). Although some phenotypic switches can be induced by onies that switched from a uffy to a smooth morphology. The a change in environment (7), others may arise stochastically, smooth state of these isolates was clonally heritable and stable possibly as a bet-hedging strategy that increases the probability through multiple cell divisions, as evidenced by the formation of smooth colonies from single cells isolated from 4-d-old smooth that a subpopulation of genetically isogenic cells survive a wider colonies (Fig. 1A). However, when grown for longer periods of range of environmental perturbations (4, 8, 9). – “ ” The ability to switch phenotypes could prove especially useful time (10 16 d), blebs appeared on the colony surface of otherwise smooth colonies (Fig. 1B). We hypothesized that these for microorganisms that are unable to physically relocate to patches of cells had adopted characteristics distinct from the an environment of choice and need to weather environmental neighboring cells, and indeed, a portion of single cells isolated fluctuations. Indeed, microbes are known to adopt multiple from these patches had regained the ability to form fluffy colonies phenotypic states, and though the nature of these states can vary widely, they are all characterized by rates of switching too high to be that of spontaneous mutation (2, 3). Several interesting Author contributions: Z.T., M.H., G.A.C., E.W.J., A. Skupin, and A.M.D. designed research; examples have been discovered in opportunistic fungal patho- Z.T., M.H., E.W.J., A.C.S., V.A., and A. Sirr performed research; G.A.C., A.C.S., V.A., and A. Sirr gens. Perhaps the best characterized state switch in fungi is the contributed new reagents/analytic tools; Z.T., M.H., G.A.C., A.C.S., and A. Skupin analyzed white-opaque switch of the Candida albicans (10), which is reg- data; and Z.T., M.H., and A.M.D. wrote the paper. ulated by transcriptional feedback loops (11) and is associated The authors declare no conflict of interest. with differential virulence (12), host colonization (13) and mat- This article is a PNAS Direct Submission. ing competency (14). C. albicans can also differentiate a sub- Freely available online through the PNAS open access option. “ ” population of persister cells that, like their bacterial counterparts, Data deposition: Raw restriction-site associated DNA tag sequencing reads of strains for exhibit increased resistance to antimicrobial drugs (15). Candida which ploidy was analyzed are deposited in the National Center for Biotechnology In- glabrata exhibits at least two spontaneous and reversible phe- formation Sequence Read Archive, www.ncbi.nlm.nih.gov/sra/ (accession no. ERP002462). notypic switches: a “core switching system” and an “irregular 1Z.T. and M.H. contributed equally to this work. wrinkle switching system” (16) which exhibit differential tissue 2To whom correspondence should be addressed. E-mail: [email protected]. colonization and clearing rates in vivo (17). Cryptococcus This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. neoformans switches between smooth, mucoid, pseudohyphal, 1073/pnas.1301047110/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1301047110 PNAS | July 23, 2013 | vol. 110 | no. 30 | 12367–12372 Downloaded by guest on September 28, 2021 based on ∼3,000 markers across the genome. Together with DNA content estimates determined by flow cytometry (SI Appendix, Fig. S2), RAD-seq showed that although the original fluffy strain (F45) was euploid, transition to the smooth state (F45 Smooth) was accompanied by the gain of an extra copy of chromosome XVI (Fig. 2A). Remarkably, the additional copy of chromosome XVI was lost when the strain transitioned back to the fluffy state (Fig. 2A,F45“2nd gen”), exhibiting a morphology indistinguishable from that of the original strain. To test whether this phenomenon was specific to F45, we chose a strain (YO880) from an independent genetic background that also harbored an extra copy of chromosome XVI. YO880 is a haploid strain derived from a cross between strains originating from “Evolution Canyon” in Israel and an oak tree in North America (SI Appendix, Table S1). Similar to F45, the version of YO880 containing an extra copy of chromosome XVI was smooth (Fig. 2B), but upon extended growth produced patches of fluffy colony producing cells. RAD-seq confirmed that these fluffy isolates had also lost the extra copy of chromosome XVI (Fig. 2B). Thus, in two independent genetic backgrounds, smooth and fluffy colony morphologies were correlated with chromosome XVI copy number. To determine whether chromosome XVI disomy was sufficient for the phenotypic switch, we engineered the original strain (F45) to enrich for chromosome XVI disomes, independent of their phenotype (Fig. 3). This system (32) modifies the centromere of a target chromosome so that it can be temporarily inactivated (by exposure to galactose), thereby increasing the frequency of chromosome-specific nondisjunction (33). Galac- tose-induced nondisjunction events are selected using a marker that can exist in one of two states, an intact HIS3 or an intact URA3 (32). Only cells that have obtained two copies of the target chromosome can excise HIS3 from one copy and maintain URA3 on the other, allowing them to grow on the selective medium (SC–His–Ura). In our strain background, ∼103 out of 107 ga- + + lactose-induced cells produced His Ura colonies. Because colony morphology was difficult to score on the selective medium Fig. 1. Toggling between the fluffy and smooth states. (A) Morphology (SI Appendix, Fig. S3), we randomly chose 48 colonies and development of a colony growing from a single cell of the F45 fluffy strain assayed their morphology on rich medium (YPD) (SI Appendix, and a smooth variant (F45 Smooth). (B) Development of “blebs” on the Fig. S4). All of these colonies were smooth, and RAD-seq of surface of a F45 Smooth colony. (C) Single cells isolated by micromanipula- = fi “ ” fl a representative subset (n 22) con rmed the presence of an tion from the blebs on day 16 yielded entirely uffy or smooth colonies. additional copy of chromosome XVI (SI Appendix, Fig. S5). To Scale bars are 1 mm. test whether restoration of the euploid state was sufficient to switch colonies back to the fluffy morphology, we used minimal fl with shapes indistinguishable from that of the original strain medium containing 5- uoroorotic acid (5-FOA) to select for the (Fig. 1C). We therefore set out to determine the mechanism loss of the URA3-containing copy of chromosome XVI (SI Ap- underlying this bidirectional switch. pendix, Fig. S6). In agreement with the original phenotypic selection, RAD-seq confirmed that all randomly selected colonies Aneuploidy Is Sufficient for the Phenotypic Switch. Because transi- with adequate sequence coverage (n = 12) were euploid and tion to a prion state is an epigenetic phenomenon that can affect exhibited the fluffy colony morphology (Fig. 3 and SI Appendix, colony morphology (29), we tested for the effect of prions by Fig. S5). Thus, the gain and loss of chromosome XVI is sufficient passaging the original F45 fluffy strain on guanidine hydrochlo- to reversibly toggle cells between the fluffy and smooth states. ride (SI Appendix, SI Materials and Methods), a method known to cure prions in S. cerevisiae (30), and then reassayed colony Multiple Routes to the Same Phenotype. While analyzing additional morphology on our assay medium (yeast extract/peptone/dex- fluffy to smooth isolates of F45, we were surprised to find that, in trose, YPD). The results (SI Appendix,Fig.S1) showed that the addition to chromosome XVI, several other aneuploidies altered fluffy morphology of F45 was independent of the prion status, the colony forming state of the strain. Disomies of chromosomes and therefore prions were unlikely to be the molecular mechanism III, X, and XV displayed smooth colony morphologies, and underlying the switch in our strain background. chromosome V disomy displayed an intermediate phenotype (Fig. Because changes in DNA copy number can also occur reversibly 4). As with chromosome XVI, the subsequent reversion of all four and at relatively high frequencies, we next tested our strains for disomic strains back to the original fluffy morphology was ac- aneuploidy (chromosome numbers that deviate from multiples companied by restoration of the euploid karyotype (SI Appendix, of the haploid chromosome count). Using a high-throughput Fig. S7). Thus, the ability of different disomies to trigger the bi- restriction-site associated DNA tag sequencing (RAD-seq) strat- directional toggle suggests that there are multiple, reversible egy that directs genome sequencing to specific restriction sites routes to the same phenotypic state. We note that the screen (31) thereby sequencing the same ∼1% of each strain in a highly also yielded a number of euploid smooth isolates (SI Appendix, multiplexed fashion, we determined the karyotype of our strains SI Text), suggesting that additional mechanisms may exist.

12368 | www.pnas.org/cgi/doi/10.1073/pnas.1301047110 Tan et al. Downloaded by guest on September 28, 2021 Fig. 2. Change in morphology affected by chromosome XVI copy number. (A) Phenotypic toggling of naturally derived strains. Plots of RAD-seq data show the presence of an extra chromosome XVI in F45 Smooth and its subsequent loss in the ﬂuffy revertant (F45 “2nd Gen”). Chromosomes are alternatively colored black and red. (B) Colony morphology in YO880 (SI Appendix,TableS1), a strain unrelated to F45. The loss of an extra chromosome XVI also correlates with the gain in complex colony morphology in this strain background.

Switch Modulated by Gene-Specific Copy Number Variation. Our a plasmid containing seven full-length genes that was able to results are consistent with either of two models. First, because confer the smooth state (SI Appendix,Fig.S9A). We subsequently numerous biological processes are required to form a fluffy determined that this effect was due to the increased copy number colony (26–28), these five chromosomes could each contain of a single gene DIG1 (Fig. 6B and SI Appendix, SI Text). DIG1 dosage sensitive genes necessary for the function or regulation of encodes a known repressor of the MAPK pathway (35), and our key processes. Alternatively, the fluffy state could be sensitive to results are consistent with a model in which overexpression of some more general aspect of aneuploidy, such as the DNA DIG1 leads to the down-regulation of the fluffy trait (26). How- content of the cell. To test whether any chromosomal aneuploidy ever, the intermediate phenotype obtained by restoring DIG1 copy could confer the smooth morphology, we introduced conditional number in the context of the XVI disomy (SI Appendix,Fig.S9B) GENETICS centromere constructs onto the 11 remaining chromosomes (I, suggests that additional genes on chromosome XVI may con- II, IV, VI, VII, VIII, IX, XI, XII, XIII, and XIV) and selected tribute to the phenotypic switch. for each disomy (SI Appendix, Fig. S8 and Table S2). Of the 11 disomies selected, four were either too sick or unstable to assay (chromosome II, VI, XI, and XIII) and two could only be stably recovered in conjunction with additional aneuploidies (disome IV paired with VII and disome XII paired with XV) (SI Ap- pendix, SI Text). This last set of strains containing multiple disomies suggested that genetic interactions may play a role in the formation of colony morphology. For example, whereas strains disomic for chromosome III were smooth, and strains disomic for chromosome IX were fluffy, strains disomic for both III and IX were smooth (Fig. 5). However, of the five chromosomes that could be stably recovered as individual disomies, only one (chromosome VII) yielded smooth colonies through day 4 of growth, whereas the remaining four disomies (chromosome I, VIII, IX, and XIV) were all fluffy at the same time point (Fig. 5). The fact that several disomies, spanning the continuum of chromosome sizes, failed to trigger the smooth transition strongly supports the hypothesis that the switch is due to specific gene effects rather than general DNA content changes. To test whether this phenotypic toggle is in fact modulated by Fig. 3. Phenotypic toggling of the strain containing a conditional centro- the copy number variation of specific genes, we transformed the mere on chromosome XVI (F45cond.XVI). The protocol used to select chro- original F45 (fluffy) strain with a set of low copy plasmids con- mosome gain and loss is depicted (right). Scale bar is 1 mm. Images were taining portions of chromosome XVI (34). This screen identified taken on day 4 of colony growth.

Tan et al. PNAS | July 23, 2013 | vol. 110 | no. 30 | 12369 Downloaded by guest on September 28, 2021 liquid medium the smooth, aneuploid strain showed only a mod- est decrease in growth rate and even a statistically significant increase in cell yield at saturation (Fig. 6E). These results suggest that the smooth state has advantages for growth in liquid media even in the presence of the presumed growth disadvantage con- ferred by aneuploidy. Because the strain overexpressing DIG1 exhibits a smooth colony morphology (Fig. 6B) without the burden of an additional chromosome, the smooth state is genetically separable from the aneuploid state. We compared the growth characteristics of F45 transformed with either a low copy plasmid containing DIG1 or an empty vector. In these experiments the fluffy (vector alone) strain also exhibited a growth advantage over the smooth (DIG1) strain on solid media (Fig. 6D), whereas in liquid medium the DIG1-mediated smooth strain exhibited both a higher maximal growth rate and cell yield (Fig. 6F). Thus, when the cost of aneuploidy is removed, the smooth state shows clear growth advantages in liquid. Discussion The phenotypic changes brought about by aneuploidy can arise from a general increase in DNA content, dosage changes in fi Fig. 4. Multiple disomies induce a similar phenotype. Colony morphology speci c gene products, or a combination of both factors. In of the different disomic yeast strains obtained by phenotypic screening. Plots S. cerevisiae some traits, such as decreased growth rates and G1 show the presence of the extra chromosomes identified by RAD-seq. Scale cell cycle delays, appear to be general effects of aneuploidy (37), bar is 1 mm. Images were taken on day 4 of colony growth. whereas others, such as altered drug resistance and protein abundance, have been linked to the duplication of specific chromosomes or genes (38). Our results add a complex, multi- Growth Advantages of the Two States. Phenotypic switching sug- cellular phenotype to the set of traits known to be regulated by gests that the states may exhibit distinct fitness landscapes under aneuploidy. Cells in fluffy colonies organize to form intricate different environmental conditions. Because the organization of systems of hollow channels that increase the distances between cells into the fluffy colony structure is a solid media phenomenon cells, allowing for more rapid colonization of surfaces and greater and because previous work has shown that fluffy colonies contain acquisition of resources (21). At the same time, they perform more cells than smooth colonies (36), we chose to compare the metabolically intensive processes, such as the production of ex- growth characteristics of smooth and fluffy colony forming tracellular matrix, that while aiding the protection of the colony, strains in both liquid and solid media (SI Appendix, SI Materials require significant nutrient and energy expenditure (21). Switching and Methods). We first compared the original fluffy euploid (F45) to the smooth state when such protection is not needed has been and the smooth XVI disome (F45 Smooth). On solid medium, proposed as an energy conservation strategy (39). Our results the fluffy strain had a substantial growth advantage, as measured suggest that the copy number imbalance of specific chromosomes by the maximal growth rate achieved and number of cells pro- is one means by which cells can affect this state change, pre- duced by day 4 of growth (Fig. 6C). Although these results are sumably by altering the dosage of a subset of genes required for consistent with previous comparisons of cell number in fluffy and the regulation or execution of these biological processes. smooth colonies (36), they could also merely reflect decreased The surprisingly large number of chromosomes able to mod- growth rates resulting from aneuploidy (37). Interestingly, in ulate the phenotypic toggle between fluffy and smooth may be

Fig. 5. Colony morphology for disomic strains. Morphologies of representative examples of each karyotype are shown on YPD agar. All strains in this ﬁgure were rekaryotyped following micromanipulation. Only stable, reproducible karyotypes are depicted. See SI Appendix, SI Text details on sick or unstable disomies (II, VI, XI, and XIII). Images were taken on day 4 of colony growth.

12370 | www.pnas.org/cgi/doi/10.1073/pnas.1301047110 Tan et al. Downloaded by guest on September 28, 2021 for the development of novel antifungal and cancer therapeutics (44). The complexity of the problem is compounded by the fact that the genome sequence (genetic background) of an individual has the potential to affect not only the traits displayed by aneuploid cells, but also how well specific aneuploidies are toler- ated. Differences in the ability to recover specific disomes between our strain and one used in a previous study (37) may be one such example. Phenotypic switching allows organisms to access multiple states with different growth or survival advantages. Here, we examine a system in which S. cerevisiae exists in two distinct multicellular states (fluffy and smooth) and is at times able to form heterogeneous populations containing both (Fig. 1B). Be- cause the molecular mechanism underlying the fluffy-smooth switch is the mitotic gain and loss of intact chromosomes, it is likely the result of a stochastic process, although we have not directly tested this hypothesis. Furthermore, the fact that these two states exhibit different relative fitness profiles under different environmental conditions (solid and liquid media), suggests that the organism may be leveraging the stochasticity of chromosome missegregation as a bet hedging strategy. Although we identified differences in growth, the two states could easily exhibit different advantages with respect to other traits, such as longevity, resistance to desiccation, and ease of dispersal, which may prove equally or even more beneficial to the organism in the natural environment. The importance of maintaining two states and the ability of the cells to switch between them is further supported by the recent identification a prion-based switch that can modulate colony morphology changes in other strains of S. cerevisiae (29). The random gain and loss of complete chromosomes has many favorable properties as a mechanism for phenotypic switching, both in the context of a preemptive bet-hedging strategy as well as a rapid means for coping with unfamiliar environmental stresses (46). Although gene expression patterns can modulate phenotypic states (11), aneuploidy provides a more direct mode of inheritance. Additionally, because regulatory networks involve complex sets of molecular interactions that must evolve over time, aneuploidy might be a useful bet-hedging strategy for responding to environmental perturbations that are not fre- quently encountered. As aneuploidy perturbs hundreds of genes simultaneously, it has the potential to affect larger numbers of Fig. 6. Growth characteristics of fluffy and smooth strains. (A and B) Rep- genes and processes per event relative to a spontaneous muta- resentative colonies of F45 and F45 Smooth growing on YPD agar (A), F45 + tion. Importantly, unlike a spontaneous mutation, aneuploidy GENETICS vector and F45 + DIG1 growing on YPD + G418 agar as plated in a “check- and any associated growth defects can also revert at a high fre- erboard” pattern (B). Images were taken on day 4 of colony growth. (C) quency. This reversibility makes aneuploidy a particularly useful −4 Maximal growth rate (P < 10 ; n = 12 or 14 colonies; Wilcoxon rank sum mechanism in conditions that are encountered infrequently and < −7 = test) and cells per colony (P 10 ; n 20 colonies; Student t test) of F45 and are likely to change. The inherent properties of aneuploidy thus F45 Smooth grown on YPD agar. Error bars are SEM. (D) Maximal growth first allow organisms to rapidly and extensively survey the fitness rate (P = 0.025; n = 7 or 10 colonies; Wilcoxon rank sum test) and cells per < −3 = + + landscape imposed by new environmental conditions. Aneu- colony (P 10 ; n 20 colonies; Student t test) of F45 vector and F45 fl DIG1 grown on YPD + G418 agar. Error bars are SEM. (E) Maximal growth ploidy then allows organisms the exibility of reverting to their −5 −5 fi rates (P < 10 ; n = 28 or 24 wells; Student t test) and saturation OD (P < 10 ; previous state if conditions return, or stability as they ne-tune n = 28 or 24 wells; Student t test) of F45 and F45 Smooth grown in liquid YPD. their strategy to over-express only the subset of genes responsible Error bars are SEM and are <1% of the values obtained. (F)Maximalgrowth for the growth advantage if conditions remain stable (46). Thus, rates (P < 10−5; n = 14 or 27 wells; Student t test) and saturation OD (P < 10−5; aneuploidy is surprisingly well suited to provide a relatively n = 14 or 27 wells; Student t test) of F45 + vector and F45 + DIG1 grown in quick, heritable, and reversible mechanism through which cells liquid YPD + G418. Error bars are SEM and are <5% of the values obtained. and organisms can toggle between phenotypes while searching for optimal fitness solutions. proportional to the relatively large number of pathways required Materials and Methods fl – for the uffy morphology (26 28) or may indicate that the trait is Yeast Strains, Media, and Manipulation. Unless noted, standard media and particularly sensitive to changes in gene dosage. In either case, methods were used for growth and genetic manipulation of yeast (47). Strains the fact that aneuploidy provides cells multiple routes to the used in this study are listed in SI Appendix, Table S1. Colony morphology was same phenotypic endpoint has fundamental implications for assayed on YPD (2% glucose, wt/vol) plates. Respiratory competency was understanding how organisms respond to environmental per- assayed by growth on YPG (3% glycerol, vol/vol) plates. The conditional turbations. Because aneuploidy is strongly correlated with drug centromere experiments used minimal medium (SC-Ura-His, Sunrise Science – Products) to enrich for disomes and 5-FOA resistance (SC, 1 g/L 5-FOA, Zymo resistance in pathogenic microorganisms (40 43), rampant in Research) to select euploid revertants. To ensure consistency in colony cancer (44), and associated with high levels of cancer relapse after growth, all images of yeast colonies presented are from colonies arrayed into drug treatment (45), aneuploidy itself could be an important target an ordered grid either by micromanipulation with 10-mm colony separation

Tan et al. PNAS | July 23, 2013 | vol. 110 | no. 30 | 12371 Downloaded by guest on September 28, 2021 (Figs. 1–5) or FACS sorting (SI Appendix, SI Materials and Methods) at 12.7- Conditional Centromeres. Replacement of the native centromeres of chro- mm spacing (Fig. 6). mosomes with a conditional centromere was adapted from a protocol using a well-characterized construct (33) that has been modified further (32). Isolation of Smooth Colony Strains. To isolate strains with a smooth colony Briefly, galactose induction was performed by first growing the strains in fi phenotype, cells from a fluffy colony were grown overnight at 30 °C in liquid YP-Raf nose for 48 h at 30 °C to saturation and diluting 1:2,000 for growth × 6 YPD (2% glucose, wt/vol). Cells were then plated onto YPD solid agar at overnight at 30 °C. Cultures were then diluted to 5 10 cells per mL, split into two aliquots, and galactose (1.5% wt/vol final concentration) was added a density of 200 cells per plate. Colonies were grown for 3 d at 30 °C and to one. Cultures were monitored by cell count until cell density had increased then screened for the smooth colony morphology. 2.5-fold (∼3–4h).1× 107 cells were then plated onto SC-His-Ura plates to select for yeast disomic for the chromosome that contains the conditional centro- Imaging. All images were taken with a Canon PowerShot SX10IS, F8.0, 1/8-s mere. In our hands, ∼1–3 × 103 colonies were recovered on each selection shutter speed, ISO80 under consistent lighting conditions, imaging distances plate. Additionally, we see a high frequency of respiratory deficient colonies and zoom. Images were cropped and scaled with Adobe Photoshop CS5.1 on our selection plates. Because respiratory competency is generally required with no further modifications to the images. for the fluffy phenotype, we checked all colonies obtained for growth on glycerol plates and only analyzed respiratory competent strains. For 5-FOA Molecular Karyotyping by RAD-seq Coverage. The molecular karyotype of each selection, 107 cells from colonies growing up on SC-His-Ura plates were resus- strain was determined by multiplexed RAD-seq (31) of yeast genomic DNA as pended in PBS and plated onto 5-FOA plates using glass beads. Plates were described (48). For each strain, the coverage ratio of each marker is com- allowed to grow for 3 d before colonies were selected for further analysis. An 2 pared with the haploid reference and plotted against a DNA marker index average of ∼1–2 × 10 colonies were recovered on each selection plate. ordered by genomic position (chromosomes I to XVI). Marker-to-marker variation was normalized using a panel of euploid strains. Detailed descrip- ACKNOWLEDGMENTS. We thank the members of the A.M.D. laboratory, tions of calculations can be found in SI Appendix, SI Materials and Methods. J. Berman, I. Shmulevich, A. Sherman, M. Dunham, and S. Huang for helpful discussions and critical reading of the manuscript; and K. Anders and G. Prelich Raw RAD-seq reads of strains for which ploidy was analyzed have been de- for providing plasmids. This work is funded by a strategic partnership between posited in the National Center for Biotechnology Information Sequence Read the Institute for Systems Biology and the University of Luxembourg. Z.T. is Archive (www.ncbi.nlm.nih.gov/sra/) with accession no. ERP002462. funded by the Agency for Science, Technology, and Research, Singapore.

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